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TECHNISCHE UNIVERSITT MNCHEN

Lehrstuhl fr Brau und Getrnketechnologie

Barley proteins source and factor of

haze formation in beer

Elisabeth Wiesen

Vollstndiger Abdruck der von der Fakultt Wissenschaftszentrum Weihenstephan fr

Ernhrung, Landnutzung und Umwelt der Technischen Universitt Mnchen zur Erlangung

des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. H.-Chr. Langowski

Prfer der Dissertation:

1. Univ.-Prof. Dr. Th. Becker

2. Univ.-Prof. Dr. W. Back

3. Prof. Dr. E. Arendt,

University College Cork / Irland

(nur schriftliche Beurteilung)

Die Dissertation wurde am 12.10.2011 bei der Technischen Universitt Mnchen

eingereicht und durch die Fakultt Wissenschaftszentrum Weihenstephan frErnhrung, Landnutzung und Umwelt am 20.12.2011 angenommen.

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Je planmiger die Menschen vorgehen,

desto wirksamer trifft sie der Zufall.

Friedrich Drrenmatt

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Danksagung

Danksagung

Zum Abschluss einer Arbeit ist es immer schn sich zurckzuerinnern, wie alles

angefangen hat und wie viele Leute zu einem erfolgreichen Abschluss beigetragen

haben.

Meinen beiden Wegbereitern zu meiner Doktorarbeit, Prof. Becker und Prof. Back

danke ich, dass sie mir ihr Vertrauen geschenkt haben, diese Arbeit durchzufhren.

Dass sie mich gefrdert, aber auch gefordert haben und in Diskussionen mir immer

wieder neue Anregungen gegeben haben. Hier gilt mein Dank auerdem Prof.

Arendt, die mir immer mit Rat und Tat zur Seite stand und an deren Institut ich einige

Versuchsreihen durchfhren durfte. Nicht zu vergessen Prof. Langowski, der den

Vorsitz zu meiner Prfung bernommen hat.

Der Wissenschaftlichen Station fr Brauerei in Mnchen e.V. danke ich fr die

Frderung dieser Arbeit und der Weihenstephaner Jubilumsstiftung 1905 fr eine

Anschubfrderung.

Dr. Martina Gastl danke ich fr die Betreuung meiner Arbeit, fr die Zeit, die sie sich

immer fr meine Anliegen genommen hat, fr ihre Diskussionen und ihre Beitrge zu

meiner Arbeit.

Daniela Schulte danke ich fr ihre Hilfe im brokratischen TU-Dschungel, fr ihre

Geduld in allen Anliegen und fr ihre Ruhe, wenn alles drunter und drber geht.

Was aber wre eine Arbeit ohne Kollegen und Leidensgenossen. Ich danke allen

meinen Kollegen am BGT und dem ehemaligen Lehrstuhl fr Technologie der

Brauerei I fr eine gute Zusammenarbeit. Allen voran Dr. Klaus Hartmann, Dr. Stefan

Kreisz und Dr. Martin Zarnkow, deren Nachfolge ich im Bereich der

Trbungsidentifizierung und Filtrierbarkeit angetreten habe, fr die Idee zu dieser

Arbeit und fr die Organisation der Finanzierung.

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Danksagung

Alicia Muoz-Insa danke ich fr ihre Freundschaft, ihre Untersttzung in allen

Lebenslagen, ihre konstruktive Kritik zu meiner Arbeit und das Korrekturlesen meiner

Arbeit.

Meinen Brokollegen Cynthia Almaguer, Alicia Muoz-Insa, Mario Jekle, Florian

Schll und Cem Schwarz danke ich fr eine tolle Zeit.

Monika Braasch und Daria Kraus danke ich fr ihre Untersttzung im Labor und

Manuela Sailer fr ihre Hilfe bei der 2D-PAGE. Toni Pichlmeier, Rene Schneider,

Andreas Meier, Manfred Wallenwein und allen technischen Angestellten danke ich

fr ihre Untersttzung bei den Mlzungs- und Brauversuchen.

Meinen Studienarbeitern Andrea Auer, Simon Kalo, Thomas Radlmaier, Christian

Krammer, Christian Nagel, Roland Novy, Christopher Holtz, Christoph Fhr und

Christoph Neugrodda danke ich fr ihr Engagement und ihren Beitrag zum Gelingen

dieser Arbeit.

Dem ICPW danke ich fr viele interessante, konstruktive und lustige Stunden und fr

die Ehrenmitgliedschaft.

Meinen Eltern danke ich, dass sie mich immer untersttzt und gefrdert haben.

Danke auch meinen Geschwistern, dass sie immer hinter mir gestanden sind.

Meinem Mann, Hendrik Wiesen, danke ich nicht nur fr seine (Engels-)Geduld und

sein Verstndnis fr meine Arbeit, sondern auch fr das Interesse an meiner Arbeit.

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Abstract

1 Abstract

Turbidity gives the first visual impression of beer quality to the consumer. Consumers

expect from a filtered beer a clear, bright, non-hazy product which remains so during

the shelf life of the product. Hazy products are often regarded as defective and

perhaps even potentially harmful. Therefore, haze formation is an important problem

in beer production. For breweries not only costs from rejected turbid beers and

therefore an image problem arises, but also increased costs because of raised use

of filter aids have to be considered. Data from leading manufacturer of filter aids

showed that the costs of kieselgur consumption can be more than doubled in case of

filtration problems due to turbidity. According to experience in haze identification at

the Lehrstuhl fr Brau- und Getrnketechnologie, in Weihenstephan, an impact of

protein content in barley and different modified malts on haze formation directly after

filtration could be observed. This surveillance was the motivation for the intensive

study of the influence of barley proteins on haze formation in beer. This work was

accomplished with the intention to understand changes over the malting and brewing

process in protein content and composition and their influence on haze formation in

filtered beer.

This thesis therefore presents an overview of several research studies and analytical

methods on haze formation, protein analytic and haze identification. An overall

picture of the role of protein haze particles was provided. Some proteins have already

been found (protein Z, LTP1) influencing haze formation, but up to now barley

proteins have not been followed from barley into the finished beer, in their respect to

influence beer turbidity. For this reason special focus lied on changes in protein

content and composition from barley to finished beer. It was also investigated how

different malt modification changes the protein composition in finished beer and how

these differences influence final beer quality, e.g. turbidity directly after filtration.

These changes were analytically followed with global nitrogen measurement

(Kjeldahl method and determination of free amino nitrogen), a Lab-on-a-Chip

technique and 2D-PAGE. Turbidity was measured with a two angle turbidity

measurement instrument.

The first approach was to prove the existence of differences in protein composition ofbeer brewed with 100 % barley raw material to beer brewed with 100 % barley malt.

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Abstract

Differences in the protein composition of the final beer could be revealed and it could

be observed that the malting process was the reason of these differences. This was

the motivation to find the initial point of changes during malting in protein composition

in beer. The first step was a research on the influence of malting (different proteolysis

stages) on protein composition in respect to protein haze in beer.

It was possible to show simple and reproducible haze identification methods for the

brewing industry to track the source of haze formation. Differences in final beer

quality and protein composition of beer brewed with 100 % barley raw material in

comparison to beer brewed with 100 % barley malt could be shown. Subsequently

malt with different germination states was produced, to find a protein fraction which

correlates with haze formation in beer. With this experimental setup a new, not yet

identified haze forming fraction of 28 kDa was found in the beer. This fraction could

be tracked from barley over the malting process to the finished beer.

2

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Zusammenfassung

2 Zusammenfassung

Die Gewhrleistung einer konstant bleibenden Produktqualitt ber einen lngeren

Zeitraum hinweg ist eines der Hauptziele der Getrnkeindustrie. Denn Biertrinker

erwarten von einem gefilterten Bier, dass es bis zum Ende seines

Haltbarkeitsdatums seine Klarheit behlt. Trbe Biere, oder Biere die Partikel

enthalten, hinterlassen unverzglich einen negativen Eindruck, da sie den Anschein

erwecken knnen, dass eventuell sogar eine potentielle Gefhrdung gegenber des

Biergenieers besteht. Brauereien mssen nicht nur mit dem entstandenen Schaden

durch das Image-Problem kmpfen, sondern auch mit erhhten Kosten whrend der

Produktion (Filterhilfsmittel). Das Problem ist, dass selbst einwandfrei filtriertes undbiologisch sauberes Bier nach lngerer Lagerung allmhlich seinen Glanz verliert, bis

es schlielich zur Bildung einer sogenannten kolloidalen Trbung bzw. eines

Bodensatzes kommt. Dies wird vom Verbraucher nicht akzeptiert und mit einer

Qualittsminderung gleichgesetzt.

Am Lehrstuhl fr Brau- und Getrnketechnologie hat sich ber die Zeit eine

Kompetenz zur Trbungsidentifizierung entwickelt. Aufgrund von Beobachtungen

ber einen lngeren Zeitraum und Anfragen aus der Industrie, konnte festgestelltwerden, dass Trbungen insbesondere schon nach dem Filter auftreten knnen,

wenn unterschiedlich gelstes Malz verwendet wurde. Aufgrund dieser

Beobachtungen wurde in dieser Arbeit versucht, die Vernderungen der

Gerstenproteine ber den Mlzungs- und Brauprozess zu verfolgen und so deren

Einfluss auf eine Trbungsbildung schon direkt nach der Filtration festzustellen.

In dieser Doktorarbeit wurde daher ein berblick ber smtliche Forschungsarbeiten

zum Thema Trbungsbildung, Trbungsidentifizierung und Proteinanalytik gegeben.

Zustzlich wurde eine allumfassende Darstellung der Rolle von proteinischen

Partikeln in der Trbungsbildung im Bier aufgezeigt. Anhand dieser

Literaturrecherche kann gesehen werden, dass schon einige spezifische Proteine

identifiziert wurden (LTP1, Protein Z), die im Bier trbungsverursachend sind. Bis

jetzt wurde aber noch nicht versucht, Gerstenproteine ber den Mlzungs- und

Brauprozess zu verfolgen und ihren Einfluss auf die Trbungsbildung zu belegen.

Aus diesem Grund wurde, in der vorliegenden Arbeit, versucht die Unterschiede in

3

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Zusammenfassung

Proteingehalt und -zusammensetzung von der Gerste, ber das Malz, bis hin ins

fertige Bier zu erfassen.

Die Vorgehensweise zur Erfassung dieser Unterschiede war folgende. Zuerst wurden

die Unterschiede in Proteingehalt und zusammensetzung zwischen 100 %

Gerstenrohfruchtbieren und Allmalzbieren und deren Einfluss auf

Bierqualittsparameter, vor allem Trbungsneigung, untersucht. Aufgrund der

Unterschiede, vor allem in Proteingehalt und zusammensetzung, wurde

angenommen, dass vor allem der Mlzungsprozess verantwortlich fr diese

Abweichungen ist.

Daraufhin wurde Gerste bei unterschiedlichen Bedingungen (Keimtemperatur,

Weichgrad und Keimdauer) vermlzt, um aufgrund der nun entstandenen

unterschiedlichen Lsungsgrade Rckschlsse auf eine Trbungsbildung

proteinischer Ursache zu erhalten. Mit Hilfe dieses Versuchsaufbaus konnte eine

Proteinfraktion von 28 kDa gefunden werden, welche eine erhhte Trbung schon

am Filterauslauf verursacht.

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Index

3 Index

1 Abstract 12 Zusammenfassung 3

3 Index 5

4 Preamble 6

4.1 List of reviewed publications 6

4.2 List of conferences 6

4.3 Thesis Organization&Directions 8

5 Introduction 95.1 Colloids and Turbidity 9

5.2 Protein structure and function from barley to beer 14

6 Motivation 18

7 References 19

8 Summary of results 22

8.1 Protein changes during malting and brewing with focus on haze and

foam formation: a review 228.2 A critical review of protein assays and further aspects of new methods

in brewing science 37

8.3 Turbidity and haze formation in beer insight and overview 43

8.4 Comparison of beer quality attributes between 100% barley malt and

barley adjunct beer focusing on changes in the protein composition 53

8.5 Influence of the malting parameters on the haze formation of beer after

Filtration 659 Conclusion and Outlook 77

10 Appendix 80

10.1 Table of figures 80

11 Curriculum Vitae 81

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Preamble

4 Preamble

4.1 List of Reviewed Publications

(1) Steiner, E., Gastl, M., Becker, T., 2011. Protein changes during malting and

brewing with focus on haze and foam formation: a review. Eur. Food Res. Technol.

232, 191-204.

(2) Steiner, E., Back, W., 2009. A critical review of protein assays and further aspects

of new methods in brewing science. Brewing Sci. 62, 90-94.

(3) Steiner, E., Becker, T., Gastl, M.: Turbidity and Haze Formation in Beer Insightand Overview. J. Inst. Brew. 116 (4), 360368, 2010

(4) Steiner, E., Auer, A., Becker, T., Gastl, M.: Comparison of beer quality attributes

between 100% barley malt and barley adjunct beer focusing on changes in the

protein composition. Journal of the Science of Food and Agriculture, 2011 (published

online, Oct. 3rd

(5) Steiner E, Arendt EK, Gastl M, Becker T. Influence of the malting parameters on

the haze formation of beer after filtration. Eur Food Res Technol. 2011; 233 (4): 587-

97.

2011).

4.2 List of Conferences

(1) Steiner, E., Klose, C., Back, W., Arendt, E.K.: Modification of Proteins during

Malting and Brewing and their Influence on Filterability; First International Symposium

for Young Scientistst and Technologists in Malting, Brewing and Distilling, 2008, Cork

(2) Steiner, E., Becker, T., Gastl, M.: Turbidity and Haze Formation in Beer Insight

and Overview; Second International Symposium for Young Scientists and

Technologists in Malting, Brewing and Distilling 2010, Freising

(3) Steiner, E., Arendt, E.K., Becker, T., Gastl, M.: Impact of different malting

parameters on the protein composition of malt, wort and finished beer; 2010 MBAA

Convention, 2010, Providence, RI

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Preamble

(4) Steiner, E., Auer, A., Gastl, M., Kreisz, S.: Comparison of beer quality attributes

between 100% barley malt and barley adjunct beer focusing on changes in the

protein composition; 2010 MBAA Convention, 2010, Providence, RI

(5) Steiner, E., Novy, R., Gastl, M., Becker, T.: Influence of silica sol on beer quality

parameters. 33rd Congress European Brewery Convention, 2011

(6) Gastl, M., Steiner, E.; Munoz, A., Becker, T., Identification of barley varieties by

Lab-on-a-Chip capillary gel electrophoresis. MBAA Annual Conference, 2011

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Preamble

4.3 Thesis Organization & Directions

This thesis is divided into three coherent chapters. Chapter 1 is an introduction which

overviews source, formation and main components of beer haze focusing on proteinhaze. The introduction describes the necessity of this thesis referring to a solid

literature research.

Chapter 2 lists the research carried out in this PhD-thesis generated by a number of

papers accepted and published in peer-reviewed international journals. This chapter

starts with an introduction in beer proteomics (paper 1; Steiner, E., Gastl, M., Becker,

T., 2011. Protein changes during malting and brewing with focus on haze and foam

formation: a review. Eur. Food Res. Technol. 232, 191-204.). Followed by a registerof analyses methods in proteomics (paper 2; Steiner, E., Back, W., 2009. A critical

review of protein assays and further aspects of new methods in brewing science.

Brewing Sci. 62, 90-94.). Also an overview of haze identification methods is given

(paper 3; Steiner, E., Becker, T., Gastl, M.: Turbidity and Haze Formation in Beer

Insight and Overview. J. Inst. Brew. 116(4), 360368, 2010).

The two research papers (Steiner, E., Auer, A., Becker, T., Gastl, M.: Comparison of

beer quality attributes between 100% barley malt and barley adjunct beer focusing on

changes in the protein composition. Journal of the Science of Food and Agriculture,

2011; and Steiner, E., Arendt, E.K., Gastl, M., Becker, T.: Influence of the malting

parameters on the haze formation of beer after filtration. Eur. Food Res. Technol.

show the results generated in this research.

Chapter 3 discusses the overall intention of this thesis in respect to the given results

and gives a perspective on research which needs further enhancements and

overworking.

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Introduction

5 Introduction

5.1 Colloids and turbidi ty

During brewing proteins and macromolecules from raw materials undergo several

changes. Throughout mashing proteins are solubilized and transferred into the

produced wort; in wort boiling proteins are glycated and coagulated and during

fermentation and maturation process, proteins aggregate as well, because of low

pH (1).

Proteins in beer appear as colloids and are able to cause turbidity in the final product.

Therefore it is necessary to understand the influence of the brewing process and thechanges proteins are exposed to respectively also the forces which influence particle

aggregation. In beer turbidity appears either directly after filtration or after some time

in the bottled/filled beer. The turbidity which occurs directly after filtration is linked to a

poor filtration (2) and the beer, where haze shows after some time, is referred to as

colloidal instable (3-4).

Microscopic particles of one phase dispersed in another are generally called colloidalsolutions or dispersions. Most of the industrial produced foodstuffs contain colloids,

which determine their rheological property and texture. Colloids are particles within a

size range from few nanometers up to some microns and are able to exist between

all possible states of aggregation (e.g. aerosols or emulsions) (5).

The term colloid is derived from the Greek word kolla for glue. It was originally

used for gelatinous polymer colloids, which were identified by Thomas Graham in

1860 in experiments on osmosis and diffusion (6).

Colloids are defined as follows:

The term colloidal refers to a state of subdivision, implying that the molecules or

polymolecular particles dispersed in a medium have at least in one direction a

dimension roughly between 1 nm and 1 m, or that in a system discontinuities are

found at distances of that order The name dispersed phase for the particles should

be used only if they have essentially the properties of a bulk phase of the same

composition... A fluid colloidal system composed of two or more components may be

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Introduction

called a sol, e.g. a protein solWhen a sol is colloidally unstable (i.e. the rate of

aggregation is not negligible) the formation of aggregates is called coagulation or

flocculation... The rate of aggregation is in general determined by the frequency of

collisions and the probability of cohesion during collision. (7).

Colloids are aggregations of small molecules due to the delicate balance of weak

attractive forces (such as the van der Waals force) and repulsive forces. The

aggregation depends on the physical environment, particularly the solvent. When the

solvent changes, the aggregation may collapse (8).

In solutions particles are exposed basically to three different forces: A gravitational

force, which influences the settling/raising of particles, depending on their density

relative to the solvent; a viscous drag force, which influences the motion of the

particles and the natural kinetic energy of particles and molecules, which causes

Brownian motion (6). Colloidal particles are constantly in motion. The irregular

movement and collision of particles in liquids is due to the Brownian Motion. Colloidal

systems are solutions of large molecules, where the large molecules are the

colloidal/Brownian particles. The minimum size of a Brownian particle is about 1 nm

and the maximum about 10 m (9). The Browninan movement is described as The

movement of particles in a colloidal system such as an aerosol caused by collision

with the molecules in the fluid in which the particles are imbedded. (7). With this

movement favorable conditions for collisions between colloids can be created, which

leads to enlargement of colloids and therefore to visible particles (10). In Figure 1

size ranges of colloids, particles and other substances and their visibility for human

eyes and microscopes are illustrated (11).

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Introduction

Figure 1: Size ranges of particles, colloids and other substances (11)

There also exist several physical and chemical forces between particles which make

them combine and form larger particles (i.e. colloids). These forces can be of

different nature (12):

Adhesive forces, which are the attractive forces between different molecules, are

caused by forces acting between two substances, such as mechanical forces and

electrostatic force. Cohesive forces are intermolecular forces and exist between

molecules of the same substances. These forces are for example:

Electromagnetic forces between opposite charged ions which lead to

covalent/ionic bonds and hydrogen bonding.

The total force between polar and non-polar (but not ionic) molecules is called

the van der Waals force, which are intermolecular forces between polar

molecules (dipole-dipole). In beer (or in other aqueous solutions) these forces

arise because most materials, when dispersed in water, can be ionized to acertain degree or adsorb ions from solutions and therefore become

charged (6). Depending on the forces, which exist between macromolecules,

colloids and particles and/or between particles and the surrounding liquid,

haze is formed in beer.

To describe the turbidity of a solution (beer) on a scientific basis, turbidity

measurement is necessary. The basis for turbidity measurement of solutions is theability of particles to scatter light. In a colloidal dispersion particles exist in the size

Colloids in solution

Colloidal particles

Bacteria

Clay

Pollen

Fog

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Introduction

range from 1-1000 nm. Particles of this size exhibit a large surface area. Due to this

enlarged surface, colloids scatter light and the scattering can be calculated as

turbidity. When light goes through a colloidal solution at a 90 angle a light

scattering can be observed. This is referred to as Tyndall Effect (10). This can be

seen in Figure 2, where the propagation of light in a homogenous media (A) and in a

medium containing particles (B) is displayed (13).

Figure 2: Light propagation in a homogenous medium and a medium containing solid particles

Tyndall was the first to study the phenomenon of the scattering of light by particles incolloidal solution. In 1944-1947 Debye was the first to use light scattering (the

measurement of light-scattering intensity) to determine the molecular weight of a

macromolecule in dilute solution (8). Figure 3 shows how the intensity of scatter

varies as a function of the angle for two particle diameters (14). Small particles

(1 m) becomes lopesided.

A

B

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Introduction

Figure 3: Angle dependency of light scatter of different particle sizes

Turbidity in beer is measured via turbidity photometers which detect the light,

scattered by the sample, see also Figure 4 (15).

Figure 4: Schematic figure of light scatter

In beer mostly two angles are used. One at 25 forward scattering, which indicates

bigger particles (> 1 m) for example yeast cells, and one at 90 forward scattering

which hints to smaller colloids (< 1 m) (16). According to MEBAK (17) the

specifications for turbidity in beer are for the 25 angle: < 0.5 EBC and for the 90

angle < 1 EBC.

Incident Light

Scattered Light 90

Scattered Light 25

Transmitted Light 0

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Introduction

5.2 Protein structure and function from barley to beer

In the first half of the 19 th

centuryGerardus Mulder was investigating the properties

of substances extractable from both animal and plant tissues. He found these to

contain carbon, hydrogen, nitrogen, and oxygen and believed them to be without

doubt the most important of the known substances without them life would be

impossible on our planet Mulder named these substances proteins from the

Greek, meaning first or foremost (18)

In the previous sections the development of colloids and therefore also protein haze

in beer, has been described. Several protein functional properties, such as

emulsification, foaming, haze formation etc. are closely related to protein

solubility (19). In beer mostly simple proteins (e.g. LTP1, protein Z), in contrary to

conjugated proteins (nucleoproteins, phosphoproteins, glycoproteins,

chromoproteins, lipoproteins and membrane proteins) exist (20). These simple

proteins in beer nearly always have a function: positive such as body and mouthfeel

and foam formation and negative, such as haze formation. Proteins in beer are

derived mostly from barley and are exposed to several forces and changes through

the malting and brewing process. The changes start during seed development (21-34) and are continued during malting, mashing, wort boiling and fermentation. During

malting, barley storage proteins are partially degraded by proteinases into amino

acids and peptides that are critical for obtaining high quality malt and therefore high

quality wort and beer. During mashing proteins are solubilized and transferred into

the produced wort. Proteins are coagulated throughout wort boiling and fermentation

and therefore can be separated (3-4, 35-38). The coagulation of proteins during the

brewing process is based on the fact, that large protein molecules are sensitive totheir surrounding and undergo denaturation, which can result in coagulation when

subjected to heat, alcohol, etc.

Denaturation: The irreversible process in which the structure of a protein is

disrupted, resulting in a partial or complete loss of function. Coagulation: The

clotting or precipitation of protein in a liquid into a semisolid compound.

Both, denaturation and coagulation are irreversible (39). Several aspects of the

brewing process are affected by soluble proteins, peptides and/or released amino

acids. Figure 5 shows an extract of main external effects on the protein content and

composition of barley, malt, wort and beer.

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Introduction

Figure 5: External effects on protein content and composition i n barley, malt, wort and beer.

According to the forces to which proteins are exposed to during malting and

brewing, proteins in beer can have different conformities and therefore they can also

have different characteristics and functions in comparison to the barley proteins.

According to these changes it is possible that haze sensitive proteins are

developed. Thus it is important to know basic protein design and how the protein

structures can be influenced.

In the following abstracts biological polymers made of proteins and peptides are

described more closely. Biological polymers consist of amino acids, nucleotides, or

sugars (8). A protein is build up by amino acids which are linked by peptide bonds. A

peptide bond is an amide linkage between an amino group of one molecule and the

carboxyl group of another. A protein which exhibits catalytic activities is an enzyme

(8). Figure 6 shows the main structure levels of a protein (40). The sequence of the

amino acid residues in a protein is called the primary structure. The primary structure

defines the charge of a molecule. The secondary structure reveals the arrangement

of the chain in space, i.e. a local folding. This is a regular geometry of the segments,

and is formed as -helix and -sheet. These coiled segments (-helix and -sheet)are formed due to intramolecular forces. How the secondary structure appears

Climate impact

Soil properties

Fertilization

StorageInfestation

Barley

Germination time

Germinationtemperature

Steeping degree

Malt

Malt

Mashing regieme

Protein content

Wort

Fermentation

Maturation

Yeast strain

Yeast condition

Beer

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Introduction

depends on the bond length and bond angles of the peptide bond, the coplanar

arrangement of the atoms involved in the amide groups, the hydrogen bonds

between N-H groups and C=O groups to maintain the maximum stability, and the

range of the distance in the hydrogen bonds. The tertiary structure is, in contrary to

the secondary structure, an overall folding - a three dimensional structure. This

overall folding makes the protein compact and globular in shape. The tertiary

structure can be divided

into so called domains.

Domains are peptide

chains which can be folded

independently from the

other segments. When

domains are combined

differently, proteins with

different functions are built.

It can be said that the

function of a protein

depends on its tertiary

structure. The tertiary

structure (native

conformation) can be

denatured by forces which

cleave hydrogen bridges,

ionic or hydrophobic bonds.

Quaternary structure is the

topology of several globulararranged polypeptide

chains aggregated together

and resembles the total

protein assembly. In

contrary to tertiary structure

quaternary structure can

easily be separated by

using an external force such as ultracentrifuge. This shows that the interpeptide chain

Figure 6: Main p rotein structure levels

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Introduction

attraction is neither strong (it can easily be separated) nor weak (it sticks together to

form an assembly) (20).

A solution such as beer contains a heterogeneous mixture of proteins, i.e.: The

sample contains a wide range of molecular species. The proteins in beer can be

different in size, may have the same size, but differ in charge because of diverse

amino acid substitution. They could also be molecular homogenous and might exhibit

conformational heterogeneity. It can therefore be stated that all proteins are

polyampholytes and carry an electric charge, which is determined by the amino acid

composition, N- and C-terminal amino acids, pH, ionic strength, any post translational

changes and the nature of the buffer ions (41). The point at which the charge of the

protein is zero is called the isoelectric point. This point serves as characteristic for

every protein. Proteins precipitate easily at the isoelectric point which can also be

used for protein characterization (42-43). The fact that protein precipitate easily at the

isoelectric point is important for haze formation in beer.

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Motivation

6 Motivation

As it is described in the section introduction proteins are known to have an influence

on turbidity in final beer. From experience in haze identification and requests from theindustry it is known that not only colloidal stability but also the, until now, rather

neglected turbidity directly after filtration is an issue regarding beer quality. In the

knowledge of haze identification it was already apparent that poor malt quality and/or

over modified malt could lead to increased protein turbidity after filtration.

Many studies have been conducted on colloidal haze, but no research has been

carried out concerning protein haze directly after filtration and on the influence of

different malt parameters (i.e. time, temperature, and steeping degree). Since

experience showed influence of different malt quality on protein haze after filtration, a

literature research was conducted regarding the influence of variation in proteolysis in

malt. No studies have been found about the influence of different proteolytic modified

malt (under-, over modified malt) on protein composition in final beer. According to

these practical investigations the influence of the malting process on the influence of

protein composition in the final beer has been taken as initial point for investigations.

To get a fundamental overview on barley proteins and their influence on haze

formation in beer, the already well known barley proteome was followed during the

malting and brewing process. To gain an overall perception of the influence of barley

proteins not only different proteolysis stages were observed but also the influence of

malting itself in comparison to barley raw material and exogenous enzymes has been

investigated. This thesis deals with the influence of different malting parameters and

therefore different malting stages on final protein composition and thus on haze

formation in final beer, after filtration.

The overall purpose of this study was to identify proteins/protein fractions and to track

their origin from barley raw material into the final beer according to the haze

formation process.

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References

7 References

1. Steiner E, Gastl M, Becker T. Protein changes during malting and brewing withfocus on haze and foam formation: a review. Eur Food Res Technol.2011;232(2):191-204.2. Kreisz S. Der Einfluss von Polysacchariden aus Malz, Hefe und Bakterien aufdie Filtrierbarkeit von Wrze und Bier. Freising: TU-Mnchen; 2002.3. Kunze W. Technologie Brauer und Mlzer: VLB Berlin; 2007.4. Narzi L. Abriss der Bierbrauerei: Wiley VCH; 2005.5. Norde W. Colloids and interfaces in life sciences: CRC Press; 2003.6. Pashley RM, Karaman ME, Wiley J. Applied colloid and surface chemistry:Wiley Online Library; 2004.7. IUPAC; International Union of Pure and Applied Chemistry [database on the

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2003.13. Scattered Light [database on the Internet]. Sigrist-Photometer AG, CH-6373Ennetbrgen. 2011 [cited 15.08.2011]. Available from:http://www.photometer.com/en/abc/show.html?q=Scattered%20light.14. Scatter angle [database on the Internet]. Sigrist-Photometer AG, CH-6373Ennetbrgen. 2011 [cited 15.08.2011]. Available from:http://www.photometer.com/en/abc/show.html?q=Streuwinkel.15. Anton Paar [database on the Internet]. Anton Paar GmbH - AUSTRIA, AntonPaar Strae 20, 8054 Graz - sterreich. 2011. Available from: http://www.anton-paar.com/Web/Document/download/16866?clng=en.16. Esslinger HM, Editor. Handbook Of Brewing: Processes, Technology,

Markets2009.17. MEBAK. Brautechnische Analysenmethoden. 4th Edition ed:Methodensammlung der Mitteleuropischen Brautechnischen Analysenkommission;2002.18. Stenesh J. Biochemistry. Press P, editor. New York: Wiley Online Library;1999.19. Tiwari BK. Pulse Foods: Processing, Quality and Nutraceutical Applications:Academic Press; 2011.20. Belitz HD, Grosch W, Schieberle P. Lehrbuch der Lebensmittelchemie:Springer; 2001.21. Finnie C, Bak-Jensen KS, Laugesen S, Roepstorff P, Svensson B. Differentialappearance of isoforms and cultivar variation in protein temporal profiles revealed inthe maturing barley grain proteome. Plant Science (Amsterdam, Netherlands).2005;170(4):808-21.

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24. Grg A, Postel W, Baumer M, Weiss W. Two-dimensional polyacrylamide gelelectrophoresis, with immobilized pH gradients in the first dimension, of barley seedproteins: discrimination of cultivars with different malting grades. Electrophoresis.1992;13(4):192-203.25. Rahman S, Kreis M, Forde BG, Shewry PR, Miflin BJ. Hordein-geneexpression during development of the barley (Hordeum vulgare) endosperm.Biochem J. 1984;223(2):315-22.26. Weiss W, Postel W, Goerg A. Qualitative and quantitative changes in barleyseed protein patterns during the malting process analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with respect to malting quality. Electrophoresis.1992;13(9-10):787-97.

27. Bak-Jensen K. S., Laugesen S, Roepstorff P, Svensson B. Two-dimensionalgel electrophoresis pattern (pH 6-11) and identification of water-soluble barley seedand malt proteins by mass spectrometry. Proteomics. 2004;4(3):728-42.28. Chandra GS, Proudlove MO, Baxter ED. The structure of barley endosperm -an important determinant of malt modification. J Sci Food Agric. 1999;79(1):37-46.29. Festenstein GN, Hay FC, Miflin BJ, Shewry PR. Immunochemical studies onbarley seed storage proteins. The specificity of an antibody to "C" hordein and itsreaction with prolamins from other cereals. Planta. 1984;162(6):524-31.30. Shewry PR. Barley seed proteins. Barley. 1993:131-97.31. Miflin BJ, Shewry PR. Seed storage proteins: genetics, synthesis,accumulation and protein quality. Dev Plant Soil Sci. 1981;3(Nitrogen CarbonMetab.):195-248.32. Ostergaard O, Finnie C, Laugesen S, Roepstorff P, Svensson B. Proteomeanalysis of barley seeds: Identification of major proteins from two-dimensional gels(pI 4-7). Proteomics. 2004;4(8):2437-47.33. Ostergaard O, Melchior S, Roepstorff P, Svensson B. Initial proteome analysisof mature barley seeds and malt. Proteomics. 2002;2(6):733-9.34. Witzel K, Jyothsnakumari G, Sudhakar C, Matros A, Mock H-P. QuantitativeProteome Analysis of Barley Seeds Using Ruthenium(II)-tris-(bathophenanthroline-disulphonate) Staining. Journal of Proteome Research. 2007;6(4):1325-33.35. Jones BL, Marinac LA, Fontanini D. Quantitative study of the formation of

endoproteolytic activities during malting and their stabilities to kilning. J Agric FoodChem. 2000;48(9):3898-905.36. Evans DE, Hejgaard J. The impact of malt derived proteins on beer foamquality. Part I. The effect of germination and kilning on the level of protein Z4, proteinZ7 and LTP1. J Inst Brew. 1999;105(3):159-69.37. Slack PT, Baxter ED, Wainwright T. Inhibition by hordein of starchdegradation. J Inst Brew. 1979;85(2):112-14.38. Osman AM, Coverdale SM, Onley-Watson K, Bell D, Healy P. The gel filtrationchromatographic-profiles of proteins and peptides of wort and beer: effects ofprocessing - malting, mashing, kettle boiling, fermentation and filtering. Journal of theInstitute of Brewing. 2003;109(1):41-50.

39. Brown A. Understanding food: Principles and preparation: Wadsworth Pub Co;2010.

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40. Main protein structures levels [database on the Internet]2011 [cited15.08.2011]. Available from:http://commons.wikimedia.org/wiki/File:Main_protein_structure_levels_zh.svg.41. Needleman SB. Protein sequence determination: a sourcebook of methodsand techniques: Springer; 1970.

42. Wilkins MR. Proteome research: new frontiers in functional genomics:Springer Verlag; 1997.43. Bommarius AS, Riebel BR. Biocatalysis: fundamentals and applications: VchVerlagsgesellschaft Mbh; 2004.

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Summary of results

8 Summary of Results

8.1 Protein changes during malting and brewing with focus on

haze and foam formation: a review

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R E V I E W P A P E R

Protein changes during malting and brewing with focuson haze and foam formation: a review

Elisabeth Steiner Martina Gastl Thomas Becker

Received: 17 October 2010/ Revised: 6 December 2010 / Accepted: 13 December 2010 / Published online: 5 January 2011

Springer-Verlag 2010

Abstract Beer is a complex mixture of over 450 con-

stituents and, in addition, it contains macromolecules such

as proteins, nucleic acids, polysaccharides, and lipids. In

beer, several different protein groups, originating from

barley, barley malt, and yeast, are known to influence beer

quality. Some of them play a role in foam formation and

mouthfeel, and others are known to form haze and have to

be precipitated to guarantee haze stability, since turbidity

gives a first visual impression of the quality of beer to the

consumer. These proteins are derived from the malt used

and are influenced, modified, and aggregated throughout

the whole malting and brewing process. During malting,

barley storage proteins are partially degraded by protein-

ases into amino acids and peptides that are critical for

obtaining high-quality malt and therefore high-quality wort

and beer. During mashing, proteins are solubilized and

transferred into the produced wort. Throughout wort boil-

ing proteins are glycated and coagulated being possible to

separate those coagulated proteins from the wort as hot

trub. In fermentation and maturation process, proteins

aggregate as well, because of low pH, and can be sepa-

rated. The understanding of beer protein also requires

knowledge about the barley cultivar characteristics on

barley/malt proteins, hordeins, protein Z, and LTP1. This

review summarizes the protein composition and functions

and the changes of malt proteins in beer during the malting

and brewing process. Also methods for protein identifica-

tion are described.

Keywords Proteins Barley Malt Beer Haze

formation Foam formation

Proteins in barley and malt

Barley (Hordeum vulgare L.) is a major food and animal

feed crop. It ranks fourth in area of cultivation of cereal

crops in the world. Barley is commonly used as raw

material for malting and subsequently production of beer,

where certain specifications have to be fulfilled. These

specifications are among others: germinative capacity,

protein content, sorting (kernel size), water content, kernel

abnormalities, and infestation. Malting includes the con-

trolled germination of barley in which hydrolytic enzymes

are synthesized, and the cell walls, proteins, and starch of

the endosperm are largely digested, making the grain more

friable [13]. Proteins in beer are mainly derived from the

barley used. The mature barley grain contains a spectrum

of proteins that differ in function, location, structure, and

other physical and chemical characteristics. Barley seed

tissues have different soluble protein contents and distinct

proteomes.

The three main tissues of the barley seed are the aleu-

rone layer, embryo, and starchy endosperm that account for

about 9, 4, and 87%, respectively, of the seed dry weight

[4, 5]. The level of protein in barley is an important

determinant in considering the final product quality of beer,

for example for cultivar identification or as an indication of

malting quality parameters [4], and it is influenced by soil

conditions, crop rotation, fertilization, and weather condi-

tions. For malting barley, the balance between carbohy-

drates and proteins is important, since high protein content

reduces primarily the amount of available carbohydrates.

Proteins present in barley seeds are important quality

E. Steiner (&) M. Gastl T. Becker

Lehrstuhl fur Brau- und Getranketechnologie, Technische

Universitat Munchen, Weihenstephaner Steig 20,

85354 Freising, Germany

e-mail: Elisabeth.Steiner@wzw.tum.de

123

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DOI 10.1007/s00217-010-1412-6

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determinants. During malting, barley storage proteins are

partially degraded by proteinases into amino acids and

peptides which are critical for obtaining high-quality malt

and therefore high-quality wort and beer [1, 6, 7].

Germination provides the necessary hydrolytic enzymes

to modify the grain, which are, in the case of proteins,endoproteases, and carboxypeptidases. These enzymes

degrade storage proteins, especially prolamins (hordeins)

and glutelins [8] and produce free amino acids during ger-

mination by cleavage of reserve proteins in the endosperm

[9]. According to Mikola [10], there exist five seine carb-

oxypeptidases in germinating barley, which have comple-

mentary specificities and mostly an acidic pH optimum. All

of these carboxypeptidases consist of 2 identical subunits,

each compose of two polypeptide chains, cross-linked by

disulphide bridges [9, 11, 12]. Barley malt endoproteases

(EC.3.4.21) develop multiple isoforms mainly during grain

germination and pass through kilning almost intact [8, 13].

Jones [1317] surveyed those enzymes and their behavior

during malting and mashing. Cysteine proteases (EC

3.4.22) are clearly important players in the hydrolysis of

barley proteins during malting and mashing. However, it

seems likely that they do not play as predominant a role as

was attributed to them in the past [15, 16, 1822]. It has

been found out that metalloproteases (EC 3.4.24) play a

very significant role in solubilizing proteins, especially

during mashing at pH 5.86.0 [23]. All current evidence

suggests that the serine proteases (EC 3.4.21) play little or

no direct role in the solubilization of barley storage proteins

[23, 24], even though they comprise one of the most active

enzyme forms present in malt [22]. While none of the barley

aspartic proteases (EC 3.4.23), that have been purified and

characterized, seem to be involved in hydrolyzing the seed

storage proteins, it is likely that other members of this group

do participate. Jones [17] investigated endoproteases in

malt and wort and discovered that they were inactivated at

temperatures above 60 C. Jones et al. [6] examined the

influence of the kilning process toward the endoproteolytic

activity. These enzymes were affected by heating at 68 and

85 C, during the final stages of kilning, but these changes

did not influence the overall proteolytic activity.

Other proteins are involved in protein folding, such as

protein disulfide isomerase (EC 5.3.4.1), which catalyzes

the formation of protein disulfide bridges. Due to their

heat-sensitivity, proteinases are inactivated when the tem-

perature rises above 72 C [2530]. They are almost totally

inactive within 16 min [1, 7, 13].

Summarizing the most important factors for the protein

composition, as origin in finished beer are barley cultivar

and the level of protein modification during malting, which

is judged by malt modification which is conventionally

measured in the brewing industry as the Kolbach index

(soluble nitrogen/total nitrogen*100) [31, 32].

To get an overview of the main proteins in malt and beer,

the most studied proteins are described in the next para-

graphs. Proteins can be classified pursuant to their solubil-

ity. Osborne [3337] took advantage of this fact and

developed a procedure to separate the proteins. Proteins are

divided into water-soluble (albumins), salt-soluble (globu-lins), alcohol-soluble (prolamins), and alkali-soluble

(glutelins) fractions [3436, 38, 39]. Osborne fractionation

is a relatively simple, fast, and sensitive extractionanalysis

procedure for the routine quantitation of all protein types in

cereals in relative and absolute quantities, including the

optimization of protein extraction and of quantitative

analysis by RP-HPLC. High-performance liquid chroma-

tography (or high-pressure liquid chromatography, HPLC)

is a chromatographic technique that can separate a mixture

of compounds and is used in biochemistry and analytical

chemistry to identify, quantify, and purify the individual

components of the mixture.

Not only Osborne fractionation and HPLC but also

several other methods exist to separate and identify pro-

teins in barley, malt, wort, and beer. To get an overview

over the applications of the described methods in the

review, a description follows in the next paragraphs.

Several authors [5, 3960] characterized barley and

barley malt proteins with help of 2D-PAGE. Other authors

[25, 26, 29, 30, 32, 41, 6165] used 2D-PAGE and mass

spectrometry to fingerprint the protein composition in beer

and to evaluate protein composition with regard to foam

stability and haze formation. Klose [39] followed protein

changes during malting with the help of a Lab-on-a-Chip

technique and validated the results with 2D-PAGE. Iimure

et al. [64] invented a protein map for the use in beer quality

control. This beer proteome map provides a strong detec-

tion platform for the behaviors of beer qualityrelated

proteins, like foam stability and haze formation. The

nucleotide and amino acid sequences defined by the protein

identification in the beer proteome map may have advan-

tages for barley breeding and process control for beer

brewing. The nucleotide sequences also give access to

DNA markers in barley breeding by detecting sequence

polymorphisms.

Hejgaard et al. [6673] worked with immunoelectro-

phoresis and could identify several malt and beer proteins.

Shewry et al. [54, 7478] determined several methods for

investigation of proteins in barley, malt, and beer mainly

with different electrophoresis methods. Asano et al. [62,

63] worked with size-exclusion chromatography, immu-

noelectrophoresis and SDSPAGE. Mills et al. [79] made

immunological studies of hydrophobic proteins in beer

with main focus and foam proteins. He discovered that the

most hydrophilic protein group contained the majority of

the proteinaceous material but it also comprised polypep-

tides with the least amount of tertiary structure.

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Vaag et al. [28] established a quantitative ELISA

method to identify a 17 kDa Protein and Ishibashi et al.

[80] used an ELISA technique to quantify the range offoam-active protein found in malts produced in different

geographic regions, and using different barley cultivars.

Van Nierop et al. [30] used an ELISA technique to follow

LTP1 content during the brewing process.

Osman et al. [1820] investigated the activity of endo-

proteases in barley, malt, and mash. Hence, protein degra-

dation during malting and brewing is very important for the

later beer quality (mouthfeel, foam, and haze stability). It

was suggested that estimation of the levels of degraded

hordein (the estimation of the levels of hordein degraded

during malting truly reflects the changes in proteins during

malting and can measure the difference in barley varieties

related to proteins and their degrading enzymes) during

malting is a sensitive indicator of the total proteolytic action

of proteinases as well as the degradability of the reserve

proteins. And therefore, it is possible to predict several beer

quality parameters according the total activity of all pro-

teinases and the protein modification during malting.

To obtain good results, those separation and identifica-

tion methods can be combined. Van Nierop et al. [30], for

example, used ELISA, 2D-PAGE, RP-HPLC, electrospray

mass spectrometry (ESMS), and circular dichroism (CD)

spectrophotometry to follow the changes of LTP1 before

and after boiling.

Since there exist various methods to separate and

identify proteins in this review, an overview over existent

proteins in barley, malt, wort, and beer is provided

according to only one method, which is Osborne fraction-

ation. These fractions are described more closely in the

next sections.

Barley glutelin

About 30% of barley protein is glutelin that dissolves only

in diluted alkali [54]. Glutelin is localized almost entirely

in the starchy endosperm (Fig. 1), is not broken down later

on, and passes unchanged into the spent grains [81, 82].

Glutelin is the least well-understood grain protein frac-tion. This is partly because the poor solubility of the

components has necessitated the use of extreme extraction

conditions and powerful solvents which often cause dena-

turation and even degradation (e.g., by the use of alkali) of

the proteins, rendering electrophoretic analysis difficult.

Also, because glutelin is the last fraction to be extracted, it

is frequently affected by previous treatments and contam-

inated with residual proteins from other fractions, notably

prolamins, which are incompletely extracted by classical

Osborne procedures [83]. It has not been possible to pre-

pare an undenatured glutelin fraction totally free of con-

taminating hordein [3].

Barley prolamin

The prolamin in barley is called hordein and it constitutes

about 37% of the barley protein. It dissolves in 80% alcohol

and part of it passes into spent grains. Hordein is a low-

lysine, high-proline, and high-glutamine alcohol-soluble

protein family found in barley endosperm (Fig. 1). It is the

major nitrogenous fraction of barley endosperm composing

3555% of the total nitrogen in the mature grain [1, 8486].

Hordeins are accumulated relatively late in grain develop-

ment, first being observed about 22 days after anthesis

(when the grain weighs about 33% of its final dry weight)

and increasing in amount until maximum dry weight is

reached [87]. The major storage proteins in most cereal

grains are alcohol-soluble prolamins. These are not single

components, but form a polymorphic series of polypeptides

of considerable complexity [88]. Hordein is synthesized on

the rough endoplasmic reticulum during later stages of grain

filling and deposited within vacuoles in protein bodies [89,

90]. Silva et al. [91] ascertained that the exposure of

hordeins to a proteolytic process during germination redu-

ces its content and originates in less hydrophobic peptides.

Fig. 1 Shematic longitudinal

section of a barley grain [81]

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Some malt watersoluble proteins result from the hordein

proteolysis. Hordeins are the most abundant proteins in

barley endosperm characterized by their solubility in alco-

hol. These storage proteins form a matrix around the starch

granules, and it is suggested that their degradation during

malting directly affects the availability of starch to amylo-lytic attack during mashing [92].

Shewry [75, 77] divided the hordeins according to their

size and amino acid composition in four different fractions

(A-D), dependent on their size and amino acid composi-

tion. A-hordeins (1525 kDa) seem to be no genuine

storage proteins as they contain protease inhibitors and

a-amylases. B-hordeins (3245 kDa) are rich in sulfur

content and are, with 80%, the biggest hordein fraction.

B-hordeins have a general structure, with an assumed sig-

nal peptide of 19 aminoacid residues, a central repetitive

domain rich in proline and glutamine residues, and a

C-terminal domain containing most of the cysteine residues

are encoded by a single structural locus, Hor2, located on

the short arm 1 of chromosome 1H(5), 78 cM distal to the

Hor1 locus which codes for the C-hordeins. C-hordeins

(4972 kDa) are low in sulfur content, and D-hordeins

([100 kDa) are the largest storage proteins and are enco-

ded by the Hor3 locus located on the long arm of chro-

mosome 1H(5) [85, 87, 93, 94].

Cereal prolamins are not single proteins but complex

polymorphic mixtures of polypeptides [54]. During malt-

ing, disulfide bonds are reduced and B- and D-hordeins are

broken down by proteolysis. Well-modified malt contains

less than half the amount of hordeins present in the original

barley. D-hordeins are degraded more rapidly than their

B-type counterparts, and the latter are more rapidly degraded

than C-hordeins [3, 95].

Barley albumins and globulins

Many researchers extract a combined salt-soluble protein

fraction, because water extracts contain globulins as well as

albumins. The two classes of proteins may be separated by

dialysis, but there is considerable overlap between the two

[83]. Albumins and globulins consist mainly of metabolic

proteins, at least in the cereal grains [96] and are found in

the embryo and the aleurone layer, respectively [81, 82].

Whereas prolamins are degraded during germination, al-

bumins and other soluble proteins increased during the

germination process [92].

Globulins

The globulin fraction of barley is called edestin. It dissolves

in dilute salt solutions and hence also in the mash. It forms

about 15% of the barley protein. Edestinforms 4 components

(a,b, c,and d) of which thesulfur-containingb-globulin does

notcompletely precipitate even on prolongedboiling andcan

give rise to haze in beer. Enzymes and enzyme-related pro-

teins are mainly albumins and globulins [42].

Albumins

The albumin of barley is called leucosin. It dissolves in

pure water and constitutes about 11% of the protein in

barley. During boiling, it is completely precipitated.

a-Amylase, protein Z, and lipid transfer proteins are barley

albumins and are important for the beer quality attributes:

foam stability and haze formation [97]. Albumins can be

further divided into protein Z and lipid transfer proteins as

functional proteins

Protein Z

Protein Z belongs to a family of barley serpins and consists

of at least four antigenically identical molecular forms with

isoelectric points in the range 5.555.80 (in beer:

5.15.4), but same molecular mass near 40 kDa [1, 55, 67,

68, 98]. Protein Z is hydrophobe and exists in free and

bound forms in barley, like a-amylase, and there also exist

heterodimers. Protein Z contains 2 cysteine and 20 lysine

residues per monomer molecule and is relatively rich in

leucine and other hydrophobic residues. Protein Z accounts

for 5% of the albumin fraction and more than 7% in some

high-lysine barleys [67, 99]. The content of protein Z in

barley grains depends on the level of nitrogen fertilization

[67, 100]. Protein Z makes up to 20170 mg/L of beer

protein [79]. In mature seeds, protein Z is present in thiol

bound forms, which are released during germination [101].

The function of the protein is at present unknown but it is

known that it is deposited specifically in the endosperm

responding to nitrogen fertilizer, similar to the hordein

storage proteins. The synthesis is regulated during grain

development at the transcriptional level in dependence of

the supply of nitrogen [98, 100, 102, 103]. It is stated that

upregulation of transcript levels could be effectuated

within hours, if ammonium nitrate was supplied through

the peduncle, and equally rapid reduced when the supply

was stopped [103]. Finnie et al. [49] investigated the pro-

teome of grain filling and seed maturation in barley. They

identified a group of proteins that increased gradually both

in intensity and abundance, during the entire examination

period of development and were identified as serpins. Also

Sorensen [55] and Giese [98] could detect the expression of

protein Z4 (a subform of protein Z) only during germina-

tion. Protein Z4 has an expression profile similar to

b-amylase and seed storage proteins (hordeins).

Three distinct serpin sequences from barley could be

found in the databases SWISSPROT and TREMBL: pro-

tein Z4, protein Z7, and protein Zx. These different protein

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Z forms are thought to have a role as storage proteins in

plants, due to their high Lys content and the fact that

serpin gene expression is regulated by the high-Lys

alleles lys1 and lys3a [49, 104].

Hejgaard et al.[68] suggest that the precursors of protein Z

originate from chromosomes 4 and 7, and thus,theyare namedprotein Z4 and protein Z7. Rasmussen and co-workers [105]

were able to estimate the size of protein Z mRNA at 1.800 b.

This is sufficient to code for the 46.000 or 44.000 MW pre-

cursor peptides found in vitro translations plus leave 400500

b for the 50 and 30 non-coding regions. Doll [106] and Ras-

mussen [107] suggest that protein Z could be a candidate for

modulation of the barley seed protein composition to balance

the nutritional quality of the grain. Giese and Hejgaard [98]

found out that during germination, protein Z becomes the

dominant protein in the salt-soluble fraction in developing

barley. The proteins in barley malt are known to be glycated

by D-glucose, which is a product of starch degradation during

malting [108]. Bobalova et al. [109] investigated in their

research theglycation of protein Z andfound out that protein Z

glycation is detectable from the second day of malting. The

role of protein Z in beer is described more detailed in the

sections foam and haze formation.

Lipid transfer protein

Lipid transfer proteins (LTPs) are ubiquitous plant lipid-

binding proteins that were originally identified by their

ability to catalyze the transfer of lipids between mem-

branes. LTPs are abundant soluble proteins of the aleurone

layers from barley endosperm. The compact structure of

the barley LTP1 comprises four helices stabilized by four

disulfide bonds and a well-defined C-terminal arm with no

regular secondary structure [110] which is shown in Fig. 2,

where a 3D and surface protein of barley LTP native

protein (here called 1LIP, red) is shown [111]. In com-

parison with other plant lipid transfer proteins, the barley

protein has a small hydrophobic cavity but is capable of

binding different lipids such as fatty acids and acyl-CoA

[25, 112, 113]. According to molecular mass, this multi-

gene family is subdivided into two subfamilies, ns-LTP1

(9 kDa) and ns-LTP2 (7 kDa); both located in the aleurone

layer of the cereal grain endosperm [56, 114]. LTP1 and

LTP2 are expressed in barley grain but only LTP1 has been

able to be detected in beer. LTP1 is claimed to be an

inhibitor of malt cysteine endoproteases [14, 115]. The role

of LTP1 in beer is described more detailed in the sections

foam and haze formation.

Protein Z and LTP1

Evans [116, 117] investigated the influence of the malting

process on the different protein Z types and LTP1. He

discovered that the amount of LTP1 did not change during

germination but a significant proportion of the bound/latent

protein Z was converted into the free fraction. He claims

that during germination, proteolytic cleavage in the reac-

tive site loop converts protein Z to a heat and protease

stable forms, and hence, they can survive the brewing

process. He ascertained also that kilning reduced the

amount of protein Z and LTP1 [66, 118].

Evans [116] analyzed feed and malting barley varieties

and could not find any differences in the level of protein Z

and LTP1. He also ascertained malt-derived factors that

influence beer foam stability, such as protein Z4, b-glucan,

viscosity, and Kohlbach index. Beer components (pro-

tein Z4, free amino nitrogen, b-glucan, arabinoxylan, and

viscosity) were correlated with foam stability [117]. Pro-

tein Z4, protein Z7, and LTP1 have been shown to act as

protease inhibitors [116, 119, 120].

Proteins in wort and beer

Proteins influence the whole brewing process not only in

the form of enzymes but also in combination with other

substances such as polyphenols. As enzymes, they degrade

starch, b-glucans, and proteins. In proteinprotein linkages,

they stabilize foams and are responsible for mouthfeel and

flavor stability, and in combination with polyphenols, they

are thought to form haze. As amino acids, peptides, and sal

ammoniac, they are important nitrogen sources for yeast

[121]. Only about 20% of the total grain proteins are water

soluble. Barley water-soluble proteins are believed to be

resistant to proteolysis and heat coagulation and hence pass

through the processing steps, intact or somewhat modified,

to beer [116, 122, 123]. Several aspects of the brewing

process are affected by soluble proteins, peptides, and/or

Fig. 2 3D and surface protein of barley LTP native protein (1LIP,

red) is shown [111]

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amino acids that are released. No more than one-third of

the total protein content passes into the finished beer which

is obtained throughout mainly two processes; mashing and

the wort boiling. Mashing is the first biochemical process

step of brewing and completes the enzymatic degradation

started during malting. Enzymes synthesized during malt-

ing are absolutely essential for the degradation of largemolecules during mashing. These enzymes are displayed in

Table 1 [1, 7]. The three biochemical basic processes

taking place during malting are cytolysis, proteolysis, and

amylolysis, which are indicated by b-glucan, FAN, and

extract concentration, respectively. In order to get good

brews, part of the insoluble native protein must be con-

verted into soluble protein during malting and mashing.

This fraction comprises a mixture of amino acids, peptides,

and dissolved proteins, and a major portion of it arises by

proteolysis of barley proteins [23]. During the brewing

process, there are three possibilities to discard the

(unwanted) proteinic particles. The first opportunity is

given during wort boiling, where proteins coagulate and

can be removed in the whirlpool. The second, during

fermentation, where the pH decreases and proteinic parti-

cles can be separated by sedimentation. The third step is

during maturation of beer. During maturation, proteins

adhere on the yeast and can be discarded [124].

It has also been demonstrated that yeast proteins are

present in beer, but only as minor constituents [73]. Beer

contains*500 mg/L of proteinaceous material including a

variety of polypeptides with molecular masses ranging

from\5 to[100 kDa. These polypeptides, which mainly

originate from barley proteins, are the product of the

enzymatic (proteolytic) and chemical modifications

(hydrogen bonds, Maillard reaction) that occur during

brewing, especially during mashing, where proteolytic

enzymes are liable for those modifications [125]. A beer

protein may be defined as a more or less heterogeneous

mixture of molecules containing the same core of peptidestructure, originating from only one distinct protein present

in the brewing materials [126]. Jones [1317] surveyed

proteinases and their behavior during malting and mashing.

Proteinases are not active in beer anymore; hence, they are

inactivated when the temperature rises above 72 C, which

happens already during mashing [1, 7, 13, 2530].

Proteins influence two main quality aspects in the final

beer: 1st haze formation and 2nd foam stability. In the

following lines, these quality attributes are described in a

more detailed way.

Haze formation

Proteins play a major role in beer stability; hence, they are,

beside polyphenols, part of colloidal haze. There exist two

forms of haze; cold break (chill haze) and age-related haze

[127]. Cold break haze forms at 0 C and dissolves at

higher temperatures. If cold break haze does not dissolve,

age-related haze develops, which is non-reversible. Chill

haze is formed when polypeptides and polyphenols are

bound non-covalently. Permanent haze forms in the same

manner initially, but covalent bonds soon form and

Table 1 Enzymes in barley and barley malt [1, 7, 166, 167]

Enzyme Substrate Product

Cytolysis b-glucan-solubilase Matrix linked b-glucan Soluble, high molecular weight b-glucan

Endo-b-(1-3)

glucanase

Soluble, high molecular weight b-glucan Low molecular weight b-glucan, cellobiose,

laminaribioseEndo-b-(1-4)

glucanase

Soluble, high molecular weight b-glucan Low molecular weight b-glucan, cellobiose,

laminaribiose

Exo-b-glucanase Cellobiose, laminaribiose Glucose

Xylanase Hemicellulose b-D-Xylose

Proteolysis Endopeptidase Proteins Peptides, free amino acids

Carboxypeptidase Proteins, peptides Free amino acids

Aminopeptidase Proteins, peptides Free amino acids

Dipeptidase Dipeptides Free amino acids

Amylolysis a-amylase High and low molecular weight a-glucans Melagosaccharides, oligosaccharides

b-amylase a-glucans Maltose

Maltase Maltose Glucose

Limit dextrinase Limit dextrins Dextrins

Pullulanase a-1,6-D-glucans in amylopectin, glykogen,

pullulan

Linear amylose fractions

Other Lipase Lipids, lipidhydroperoxide Glycerine, free fatty acids, fatty acid hydroperoxide

Lipoxygenase Free fatty acids Fatty acid hydroperoxide

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insoluble complexes are created which will not dissolve

when heated [128]. Proanthocyanidins (condensed tannins)

from the testa tissue (seed coat) of the barley grain are

carried from the malt into the wort and are also found after

fermentation of the wort in the beer. There they cause

precipitation of proteins and haze formation especially

after refrigeration of the beer, even if it previously had

been filtered to be brilliantly clear [129]. Proteins, as the

main cause of haze formation in beer, can be divided into

two main groups: 1st proteins and 2nd their breakdown

products. Protein breakdown products are characterized by

always being soluble in water and do not precipitate during

boiling. Finished beer contains almost only protein break-

down products [126]. The content of only 2 mg/L protein is

enough to form haze [118]. Beer contains a number of

barley proteins that are modified chemically (hydrogen

bond formation, Maillard reaction) and enzymatically

(proteolysis) during the malting and brewing processes,

which can influence final beer haze stability. Leiper et al.

[130, 131] found out that the mashing stage of brewing

affects the amount of haze-active protein in beer. If a beer

has been brewed with a protein rest (4852 C), it may

contain less total protein but more haze-active proteins

because the extra proteolysis caused release of more haze

causing polypeptides. Asano et al. [62] investigated dif-

ferent protein fractions and split them in 3 categories: 1st

high, 2nd middle and 3rd low molecular weight fractions

being high molecular weight fractions:[40 kDa, middle

molecular weight fraction: 1540 kDa and low molecular

weight fraction:\15 kDa. Nummi et al. [132] even sug-

gested that acidic proteins derived from albumins and

globulins of barley are responsible for chill haze formation

(Table 2).

Researchers proofed that proline-rich proteins are

involved in haze formation [63, 65, 124, 127, 128, 130, 131,

133137]. Outtrup et al. [138] say that haze-active proteins

are known to be dependent on the distribution of proline

within the protein. Nadzeyka et al. [127] suggested that

proteins in the size range between 1535 kDa comprised the

highest amount of proline. It was also investigated that

proline and glutamic acid-rich hordeins, in the size range

between 1030 kDa, are the main initiators causing haze

development [63, 74]. b-Amylase, protein Z, and two chy-

motrypsin inhibitors have relatively high-lysine contents

[100]. Barley storage proteins that are available for hydro-

lysis are all proline-rich proteins [15]. Dadic and Belleau

[139, 140] on contrary say that there is no specific amino

acid composition for haze-active proteins. Leiper [130, 131]

even says that not only the mainly consistence of proline and

glutamic acid of the glycoproteins is responsible for causinghaze but also that the carbohydrate component consists

largely of hexose. It was found out that the most important

glycoproteins for haze formation are 16.5 and 30.7 kDa in

size. Glycation is a common form of non-enzymatic modi-

fication that influences the properties of proteins [109]. Non-

enzymatic glycation of lysine or arginine residues is due to

the chemical reactions in proteins, which happen during the

Maillard reaction [109]. It is one of the most widely spread

side-chain-specific modifications formed by the reaction of

a-oxoaldehydes, reducing carbohydrates or their derivatives

with free amine groups in peptides and proteins, such as

e-amino groups in lysine and guanidine groups in arginine

[141, 142]. The proteins in barley malt are known to be

glycated by D-glucose, which is a product of starch degra-

dation during malting [108]. D-glucose reacts with a free

amine group yielding a Schiff base, which undergoes a rapid

rearrangement forming more stable Amadori compounds.

Haze-sensitive proteins

Polypeptides that are involved in haze formation are also

known as sensitive proteins. They will precipitate with

tannic acid, which provides a mean to determine their

levels in beer. Proline sites of these polypeptides bind to

silica gel hydroxyl groups so that haze-forming proteins are

selectively adsorbed, since foam proteins contain little

proline and are thus not affected by silica treatment [143].

Removal of haze forming tannoids can be effected using

PVPP [143]. To assure colloidal stability, it is not neces-

sary to remove all of the sensitive proteins or tannoids.

Identification of a tolerable level of these proteins can be

used to define a beer composition at bottling that delivers

satisfactory haze stability [94, 99]. To prolong stability of

beer, stabilization aids are used. Haze-forming particles are

removed with: (a) silica, which is used to remove proline-

rich proteins that have the ability to interact with poly-

phenols to form haze in bright beer, or (b) PVPP, which is

used to remove haze-active polyphenols.

Evans et al. [144] investigated the composition of the

fractions which were absorbed by silica. This analysis

revealed that the mole percentage of proline ranged

between 33.2 and 38.0%, and of glutamate/glutamine

between 32.7 and 33.0%, consistent with the proline/glu-

taminerich composition of the hordeins [144]. Iimure

et al. [65] stated in their studies that proteins adsorbed onto

silica gel (PAS) are protein Z4, protein Z7, and trypsin/

amylase inhibitor pUP13 (TAI), rather than BDAI-1

Table 2 Distribution of hordeins in barley according to their size

[75]

Type MW (kDa) % of total hordeins

A 1016 [5

B 3046 8090C 4872 1020

D [100 [5

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(a-amylase inhibitor), CMb, and CMe. Lazaro et al. [145]

investigated the CM proteins CMa, CMb, and CMe. The

CM proteins are a group of major salt-soluble endosperm

proteins encoded by a disperse multigene family and act as

serine proteinase inhibitors. Genes CMa, CMb, and CMe

are located in chromosomes 1, 4, and 3, respectively.Protein CMe has been found to be identical with a previ-

ously described trypsin inhibitor. Furthermore, Iimure et al.

[64] analyzed proline compositions in beer proteins, PAS,

and haze proteins. It was proofed that the proline compo-

sitions of PAS were higher (ca. 20 mol%) than those in the

beer proteins (ca. 10 mol%), although those of the haze-

active proteins such as BDAI-1, CMb, and CMe were

6.68.7 mol%. These results suggest that BDAI-1, CMb,

and CMe are not predominant haze-active proteins, but

growth factors of beer colloidal haze. Serine proteinase

inhibitors have also been called trypsin/a-amylase inhibi-

tors, and it has been proposed that some of them might

inhibit the activities of barley serine proteinases. However,

none have been shown to affect barley enzymes [16].

Robinson et al. [146] identified a polymorphism for beer

haze-active proteins and surveyed by immunoblot analysis

throughout the brewing process. In this polymorphism,

some barley varieties contained a molecular weight band at

12 kDa, while in other varieties, this band was absent. Pilot

brewing trials have shown that the absence of this 12 kDa

protein conferred improved beer haze stability on the

resulting beer. This band was detected by a polyclonal

antibody raised against a haze-active, proline/glutamine

rich protein fraction; it was initially assumed that the band

was a member of the hordein protein family [144, 147].

Foam formation

Beer foam is an important quality parameter for customers.

Good foam formation and stability gives an impression of a

freshly brewed and well-tasting beer. Therefore, it is nec-

essary to investigate mechanisms that are behind foam

formation. Beer foam is characterized by its stability,

adherence to glass, and texture [148]. Foam occurs on

dispensing the beer as a result of the formation of CO2bubbles released by the reduction in pressure. The CO2bubbles collect surface-active materials as they rise. These

surface-active substances have a low surface tension, this

means that within limits they can increase their surface

area and also, after the bubbles have risen, they form an

elastic skin around the gas bubble. The greater the amount

of dissolved CO2 the more foam is formed. But foam

formation is not the same as foam stability. Foam is only

stable in the presence of these surface-active substances

[1]. Beer foam is stabilized by the interaction between

certain beer proteins, for example LTP1, and isomerized

hop a-acids, but destabilized by lipids [30, 148]. The

intention is to find a good compromise of balancing foam-

positive and foam-negative components. Foam-positive

components such as hop acids, proteins, metal ions, gas

composition (ratio of nitrogen to carbon dioxide), and gas

level, generally improve foam, when increased. Whereasfoam negatives, such as lipids, basic amino acids, ethanol,

yeast protease activity, and excessive malt modification,

decrease foam formation and stability. Free fatty acids,

which are extracted during mashing, have a negative effect

on foam stability [64, 65, 80, 85, 88, 128131, 166].

Foam-positive proteins can be divided into high

molecular weight proteins (3550 kDa) and low molecular

weight proteins (515 kDa) which primary originate from

malt but in small amount can also originate from yeast [62,

73, 148]. It is thought that during foam formation, am-

phiphile proteins surround foam cells and stabilize them by

forming a layer. They arrange themselves into bilayers, by

positioning their polar groups toward the surrounding

aqueous medium and their lipophilic chains toward the

inside of the bilayer, defining a non-polar region between

two polar ones [149]. There are two main opinions con-

cerning the nature of foaming polypeptides in beer. The

first position claims the existence of specific proteins which

basically influence foam stability. Those proteins are

known as protein Z and LTP1 [150, 151]. The second

argument claims the existence of a diversity of polypep-

tides which stabilize foam; the more hydrophobic their

nature, the more foam active they are [122, 152], like

hordeins that are rich in proline and glutamine content and

exhibit a hydrophobic b-turn-rich structure [74]. KAPP

[153] investigated the influence of albumin and hordein

fractions from barley on foam stability, because both are

able to increase the foam stability. The ability to form more

stabile foams seems to be higher by albumins than by

hordeins. Denaturation of these proteins causes an increase

in their hydrophobic character and also in their foam sta-

bility. This confirms the already known opinion that the

more hydrophobic the protein, the better is the foam sta-

bility [122, 152]. The foams from albumins are more stable

than those from hordeins. This may also be the reason for

the increased ability of albumin fractions to withstand the

presence of ethanol. The foam stability of both albumins

and hordeins is increased by bitter acids derived from hops.

Whereas the barley LTP1 does not display any foaming

properties, the corresponding beer protein is surface active.

Such an improvement is related to glycation by Maillard

reactions on malting, acylation on mashing, and structural

unfolding on brewing which was ascertained by Perrocheau

et al. [25]. During the malting and brewing processes,

LTP1 becomes a surface-active protein that concentrates in

beer foam [55]. LTP1 is modified during boiling and this

modified form influences foam stability [28, 150]. The two

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forms have been recovered in beer with marked chemical

modifications including disulfide bond reduction and rear-

rangement and especially glycation by Maillard reaction.

The glycation is heterogeneous with variable amounts of

hexose units bound to LTPs [112]. The four lysine residues

of LTP1 are the potential sites of glycation [112]. Alto-gether, glycation, lipid adduction, and unfolding should

increase the amphiphilic character of LTP1 polypeptides

and contribute to a better adsorption at airwater interfaces

and thus promote foam stability.

Van Nierop et al. [30] established that LTP1 denatur-

ation reduces its ability to act as a binding protein for foam

damaging free fatty acids and th